Characterisation of conformational changes related to the substrate specificity of coagulation factors

Factor (F) XII is a coagulation factor at the nexus of coagulation, inflammation, innate immunity, and fibrinolysis. FXII activation occurs through interactions with anionic surfaces, initiating the intrinsic pathway of coagulation and the proinflammatory kallikrein-kinin system, via activated FXI(a) and plasma kallikrein (PKa), respectively. FXII deficiency is asymptomatic of bleeding diathesis and animal models show FXIIa inhibition protects against thrombosis, making FXIIa a desirable therapeutic target. FXIIa mediated clotting in vivo is triggered by platelet secreted polyphosphates, therefore this thesis aimed to develop an experimental model to screen inhibitors under conditions representative of physiological FXII activation because global models of thrombosis under rheological conditions are scarce. The model was optimised using surface adsorbed collagen as an agonist, and was shown to be dependent on FXII, prekallikrein (PK), FXI, high-molecular-weight-kininogen (HK), and activated collagen-adhered platelets associated with phosphatidylserine exposure. This was utilised to investigate FXIa and FXa inhibitors and novel FXIIa compounds towards in vitro thrombus formation. The literature suggests an in vivo role of FXII beyond thrombus formation whereby misfolded protein aggregates trigger FXII activation without subsequent coagulation. To explore this mechanism, using advanced glycation end products of serum albumin (SA-AGE) as an exemplar misfolded protein, it was found that SA-AGE triggers purified FXII activity and separately inhibits FXIa activity and FIXa-dependent FXa generation. This was further investigated by analysing markers of intrinsic coagulation in plasma from patients with diabetes mellitus (DM). It was found that the detection of plasma AGEs was associated with higher FXIIa antigen levels, PKa activity levels, and HK degradation. These data indicate an unexpected role of AGEs in the pathophysiology of DM at the level of activation of FXII activation and its downstream consequences on inflammation and coagulation. The significance of FXIIa in DM patients remains to be explored which may provide novel therapeutic options.